Methods and principles of fixation by freeze-substitution

Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O degrees C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175 degrees C. (with precautions). The frozen tissue was placed in substituting fluid at -70 degrees C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances.